Fig. 3 |. IcsB is an 18-carbon N-fatty acyltransferase that modifies lysine residues in the C-terminal PBR of RhogTPases.

a, Mass spectrometry identification RhoA modification by IcsB in transfected 293T cells. Flag-RhoA Q63L was purified using the anti-Flag affinity resin. The total molecular mass was determined by electrospray ionization mass spectrometry. The asterisk denotes RhoA Q63L without the prenylation modification. b, Extracted ion chromatograms of Flag-RhoA Q63L purified from transfected 293T cells. Shown are graphs of the stearoylated peptide RGKKK (m/z =  441.8). c, MS/MS mass spectrum of the stearoylated peptide RGKKK in IcsB-modified Flag-RhoA Q63L purified from 293T cells. b and y fragments in (c) are obtained from the MS/MS spectra of the fatty acylated peptide (RGK(ste)KK). d, Alignment of the RhoA, Cdc42 and Rac1 C-terminal 13-residue sequences. The prenylated cysteine residues are in red, and the PBR lysines are in blue. e, In vitro reconstitution of RhoA stearoylation by IcsB. Purified farnesylated RhoA was incubated with MBP-IcsB, ³H-stearoyl-CoA and the indicated concentration of IP₆. The reactions were stopped by adding the SDS sample buffer, and subjected to SDS-PAGE followed by Coomassie blue staining and ³H autoradiography. f, Requirement of RhoA prenylation for fatty acylation by IcsB. The farnesylated or unmodified Flag-SUMO-RhoA C-terminal 10-residue (C10 aa) tail was incubated with SUMO-IcsB, ATP and cell extracts. The reactions were stopped by adding the SDS sample buffer, then subjected to 15% SDS-PAGE followed by anti-Flag immunoblotting (left). Cell lysates were also subjected to Triton X-114 partitioning (AP, aqueous phase; DP, detergent phase; right). L/S is an L193S mutation that switches the native geranylgeranylation to farnesylation. Data in a–c, e and f are representative of three independent experiments.